The long term goal of this proposal is to elucidate mechanisms responsible for the establishment and maintenance of the polarized epithelial phenotype. Epithelial cells sort plasma membrane proteins into apical and number basolateral domains in order to carry out vectorial functions for the organism. The experiments proposed will utilize state of the art imaging techniques and biochemical assays to elucidate fundamental aspects of the sorting process, protein and lipid delivery to the cell surface, and the role of the cytoskeleton. The experiments are based on recent technical advances that allow simultaneous expression of high levels of apical and basolateral markers, to study their sorting in vesicle budding assays and to follow their transport in real time in live cells. Specific aim 1 is to investigate the dual requirement for caveolins 1 and 2 in the apical targeting of GPI-anchored proteins by Fischer Rat Thyroid (FRT) cells. These cells lack both caveolins and missort model GPI-proteins to the basolateral surface; however, dual caveolin expression promotes apical delivery of GPI-proteins. Experiments will explore whether caveolins are needed to stabilize the association of GPI-proteins with specialized lipid domains termed "rafts" and to study in detail the "cycling" of cav-1 and cav-2, using temperature blocks, immunofluorescence and video microscopy. Specific aim 2 is to use morphological and biochemical techniques to study the segregation of different sets of cargo proteins at the Trans Golgi Network (TGN). TGN domains enriched in apical markers, basolateral markers or Man6P receptor will be purified using antibodies against cytoplasmic epitopes of these proteins. Specific aim 3 is to utilize these purified TGN subdomains to identify novel components of the TGN sorting/vesicle assembly machinery. In vitro vesicle budding assays and high resolution time-lapse optical microscopy will be used to study the role of known and novel TGN and cytosolic proteins (adaptors, coat proteins, motors) in the exit of cargo proteins from the TGN and in post-Golgi transport of these proteins. Specific aim 4 is to study the role of actin cytoskeleton regulatory molecules in post Golgi transport, polarized docking and establishment of the apical pole of epithelial cells. Three key proteins, cdc42, lethal giant larvae (lgl), and MacMarcks, will be investigated. The experiments proposed will provide novel information on the molecular bases of normal and abnormal function of epithelial organs, such as kidney, liver, intestine, lung and glands.